Because of emerging antibiotic resistance, there is a need to develop new antimicrobial strategies against S. aureus. Many of the virulence genes in S. aureus are often controlled by global regulatory systems. We have previously shown that the sarA locus controls the expression of many virulence determinants including surface protein adhesins and extracellular toxins. Inactivation of this locus has led to diminished rates of infection in the mutant as compared with the parent. The major sarA regulatory molecule, SarA, is a 14.5 kDa DNA binding protein that could modulate the transcription of regulatory loci (e.g. agr) as well as direct control of target genes (e.g. alpha toxin gene). Studies by Dunman and colleagues have shown that sarA could control directly and indirectly over 100 target genes. Thus, the expression of SarA must have incurred significant metabolic expenditure to the bacteria. We thus speculate that the complex sarA promoter which drives three overlapping sarA transcripts, all encoding the SarA protein, is carefully controlled by regulatory proteins at the promoter level. In the previous funding period, we have characterized the sarA promoter elements and three regulatory inputs, namely SarA, SigB and SarR, for the control of SarA expression. Importantly, the SarA protein is maximally expressed during the late exponential phase and tapered towards the postexponential phase, coinciding with the maximal expression of SarR, a repressor of SarA expression, during the postexponential phase. Contrary to data from another group, recent SarA crystal structure from our collaborator suggested that SarA is a winged helix protein, with residues R84 and R90 within the "wing" region critical to DNA binding while acidic residues D88 and E89 (part of the wing) and D8 and En (within the metal binding pocket) may be involved in effecting target gene transcription. Co-crystallization trials indicated that SarA may bind a 34-bp sequence with an inverted repeat (TGTTAAA-N19-TTTAACA). Moreover, we have found that two genes, rsr and sarZ may repress and activate sarR transcription, respectively. Predicated upon these data, we will ask three important questions: 1) Given that SarA is a winged helix protein, what is the exact binding site of SarA on the agr promoter? 2) What are the roles of D88 and E89 as well as D8 and E11 in dimer-dimer interaction and on gene transcription? 3) What roles do rsr and sarZ play in regulating the expression of SarR, a key determinant of SarA protein expression? We have proposed four major specific aims for these three questions. Our goal is to understand how SarA controls target gene and how other intracellular regulators affect SarA expression in S. aureus.